Use of vegetables for enhancing oxidative stability of omega-3 oils in the powdered state.

The preliminary study examined the effectiveness of various vegetables for the stabilisation of omega-3 oil powders against oxidative deterioration. Purees made from different vegetables (mushroom, brussels sprouts, broccoli, cauliflower, snow peas, tomato, and garlic) were employed for preparation of vegetable-tuna oil emulsions, which were subsequently freeze-dried into powders. Oxipres® data showed that vegetable-tuna oil powders had longer induction periods than neat tuna oil. During accelerated oxidation storage (40 °C/4weeks), eicosapentaenoic and docosahexaenoic acids in the vegetable-tuna oil powders were protected against oxidation, and there were lower levels of headspace secondary and tertiary oxidation products. Whole vegetable purees were suitable protective matrices for omega-3 oils. Of the various vegetable purees examined for protective effects against omega-3 oxidation, mushroom, brussels sprouts, broccoli, and cauliflower were superior to snow peas, garlic and tomato. The antioxidant properties of phytonutrients inherent in various vegetables are likely contributors to protection of omega-3 oil powders against oxidation.

Stability and partitioning of ß-carotene in whey protein emulsions during storage.

Varying the ß-carotene (0.1-0.3 g kg-1) and whey protein isolate (WPI) (2-20 g kg-1) concentrations in an oil-in-water (O/W) emulsion influenced the partitioning and stability of ß-carotene upon 30 d storage at 25 and 40 °C. The total ß-carotene in the emulsion was extracted with a solvent and quantified using UV/visible spectroscopy. The ß-carotene in oil phase was obtained using in situ Raman micro-spectroscopy. The ß-carotene in the aqueous phase was obtained by difference. Increasing ß-carotene concentration resulted in increased partitioning of ß-carotene into the aqueous phase whereas increasing WPI concentration had the opposite effect. With all freshly made emulsions, there was a higher proportion of ß-carotene found in the oil phase. At the end of the storage period, the higher proportion and concentration of ß-carotene was in the aqueous phase. This suggested that oxidation of ß-carotene occurred faster in the oil phase and that WPI in the aqueous phase protected ß-carotene against oxidation. This work informs the formulation of protein-based emulsions for the delivery of ß-carotene.

Casein polymorphism heterogeneity influences casein micelle size in milk of individual cows.

Milk samples from individual cows producing small (148-155 nm) or large (177-222 nm) casein micelles were selected to investigate the relationship between the individual casein proteins, specifically κ- and ß-casein phenotypes, and casein micelle size. Only κ-casein AA and ß-casein A1A1, A1A2 and A2A2 phenotypes were found in the large casein micelle group. Among the small micelle group, both κ-casein and ß-casein phenotypes were more diverse. κ-Casein AB was the dominant phenotype, and 3 combinations (AA, AB, and BB) were present in the small casein micelle group. A considerable mix of ß-casein phenotypes was found, including B and I variants, which were only found in the small casein micelle group. The relative amount of κ-casein to total casein was significantly higher in the small micelle group, and the nonglycosylated and glycosylated κ-casein contents were higher in the milks with small casein micelles (primarily with κ-casein AB and BB variants) compared with the large micelle group. The ratio of glycosylated to nonglycosylated κ-casein was higher in the milks with small casein micelles compared with the milks with large casein micelles. This suggests that although the amount of κ-casein (both glycosylated and nonglycosylated) is associated with micelle size, an increased proportion of glycosylated κ-casein could be a more important and favorable factor for small micelle size. This suggests that the increased spatial requirement due to addition of the glycosyl group with increasing extent of glycosylation of κ-casein is one mechanism that controls casein micelle assembly and growth. In addition, increased electrostatic repulsion due to the sialyl residues on the glycosyl group could be a contributory factor.

Structural differences between bovine A(1) and A(2) ß-casein alter micelle self-assembly and influence molecular chaperone activity.

Within each milk protein there are many individual protein variants and marked alterations to milk functionality can occur depending on the genetic variants of each protein present. Bovine A(1) and A(2) ß-casein (ß-CN) are 2 variants that contribute to differences in the gelation performance of milk. The A(1) and A(2) ß-CN variants differ by a single AA, the substitution of histidine for proline at position 67. ß-Casein not only participates in formation of the casein micelle but also forms an oligomeric micelle itself and functions as a molecular chaperone to prevent the aggregation of a wide range of proteins, including the other caseins. Micelle assembly of A(1) and A(2) ß-CN was investigated using dynamic light scattering and small-angle X-ray scattering, whereas protein functionality was assessed using fluorescence techniques and molecular chaperone assays. The A(2) ß-CN variant formed smaller micelles than A(1) ß-CN, with the monomer-micelle equilibrium of A(2) ß-CN being shifted toward the monomer. This shift most likely arose from structural differences between the 2 ß-CN variants associated with the adoption of greater polyproline-II helix in A(2) ß-CN and most likely led to enhanced chaperone activity of A(2) ß-CN compared with A(1) ß-CN. The difference in micelle assembly, and hence chaperone activity, may provide explain differences in the functionality of homozygous A(1) and A(2) milk. The results of this study highlight that substitution of even a single AA can significantly alter the properties of an intrinsically unstructured protein such as ß-CN and, in this case, may have an effect on the functionality of milk.

Decreased expression of protease inhibitor 9, a granzyme B inhibitor, in celiac disease: a potential mechanism in enterocyte destruction and villous atrophy.

The objective of this study was to assess the expression of protease inhibitor 9, a granzyme B inhibitor, in human small intestine, and to evaluate its cytoprotective role in the celiac disease of children. Twelve subjects with untreated celiac disease and thirteen healthy controls were examined by endoscopy. The expression of protease inhibitor 9 was analyzed immunohistochemically from duodenal biopsies and compared to granzyme B expression, apoptosis rate, number of intraepithelial lymphocytes and villus and crypt height data from the biopsies. We discovered that protease inhibitor 9 is expressed in the cytoplasm of the duodenal epithelial cells in the majority of cases. The enterocyte expression of protease inhibitor 9 was lower in celiac disease patients than in controls. Protease inhibitor 9 expression also showed a negative correlation with the number of apoptotic cells, overall density of granzyme B expressing intraepithelial lymphocytes, the height of the crypts and the severity of villous atrophy in duodenum. Therefore, we conclude that the protease inhibitor 9 is constantly expressed in the enterocytes of normal duodenum and the expression is decreased in celiac disease. These findings suggest that protease inhibitor 9 has a role in duodenal homeostasis and in the protection of enterocytes from misdirected granzyme B. Indeed, observed associations of lowered protease inhibitor 9 expression together with increased granzyme B expression, apoptosis rate and severity of villous atrophy suggest that impaired balance between granzyme B mediated cytotoxicity and its inhibition by protease inhibitor 9 forms an important factor in the pathogenesis of villous atrophy in celiac disease.

Enhanced creaming of milk fat globules in milk emulsions by the application of ultrasound and detection by means of optical methods.

The effects of application of ultrasonic waves to recombined milk emulsions (3.5% fat, 7% total solids) and raw milk on fat destabilization and creaming were examined. Coarse and fine recombined emulsions (D[4,3]=9.3 µm and 2.7 µm, respectively) and raw milk (D[4,3]=4.9 µm) were subjected to ultrasound for 5 min at 35°C and 400 kHz or 1.6 MHz (using a single transducer) or 400 kHz (where the emulsion was sandwiched between two transducers). Creaming, as calculated from Turbiscan measurements, was more evident in the coarse recombined emulsion and raw milk compared to that of the recombined fine emulsion. Micrographs confirmed that there was flocculation and coalescence in creamed layer of emulsion. Coalescence was confirmed by particle size measurement. These results imply that ultrasound has potential to pre-dispose fat particles in milk emulsions to creaming in standing wave systems and in systems with inhomogeneous sound distributions.

Resistant starch modification: effects on starch properties and functionality as co-encapsulant in sodium caseinate-based fish oil microcapsules.

The properties of resistant starch (RS) modified by heating starch suspensions (Heat RS) or heating followed by microfluidization (Heat-MF RS) and their functionality as co-encapsulants in sodium caseinate (NaCas) based fish oil microcapsules (50%oil:25%NaCas:25%starch) were examined. RS modification reduced molecular weight and crystallinity of the starch, with the effects being more evident for Heat-MF RS. The properties of fish oil microcapsules were influenced by the starch type used (RS, Heat RS, or Heat-MF RS) in combination with NaCas. With both physical blends and heated mixtures of NaCas and starch as encapsulants, highest encapsulation efficiency but lowest oxidative stability was obtained for the microcapsules containing Heat-MF RS. Oxidative stability was independent of heat treatment applied to the mixtures of NaCas and starch and also not related to encapsulation efficiency of the freeze-dried microcapsules. The properties of the starch used in combination with NaCas were the major determinant of the oxidative stability, with lower molecular weight and decreased crystallinity providing less protection against oxidation.

Effects of mineral salts and calcium chelating agents on the gelation of renneted skim milk.

The effects of adding CaCl2, orthophosphate, citrate, EDTA, or a mixture of these, to reconstituted skim milk (90 g of solids/kg solution) on the gelation of renneted milk were mediated by changes in Ca2+ activity and the casein micelle. At pH 6.65, the addition of citrate or EDTA, which removed more than 33% of the original colloidal calcium phosphate with the accompanying release of 20% casein from the micelle, completely inhibited gelation. Reformation of the depleted colloidal calcium phosphate and casein in the micelle, by the addition of CaCl2, removed this inhibition. When the minimum requirements for colloidal calcium phosphate and casein in the micelle were met, the coagulation time decreased with increasing Ca2+ activity, leveling off at high Ca2+ activity. The storage modulus of renneted gels, measured at 3 h, increased with increasing colloidal calcium phosphate content of micelles up to a level at which it was approximately 130% of the original colloidal calcium phosphate in the micelles. Further increases in colloidal calcium phosphate by the addition of CaCl2, orthophosphate, or mixtures of these, which did not change the proportion of casein in the micelle, decreased the storage modulus. The gelation of the renneted milk was influenced by Ca2+ activity, the amounts of colloidal calcium phosphate, and casein within the micelle, with the effects of colloidal calcium phosphate and casein within the micelle clearly dominating the storage modulus. These results are consistent with the model of Horne (Int. Dairy J. 8:171-177, 1998) which postulates that, following cleavage of the stabilizing K-casein hairs by rennet, the properties of the rennet gel are determined by the balance between the electrostatic and hydrophobic forces between casein micelles.

Progress towards single-molecule sequencing: enzymatic synthesis of nucleotide-specifically labeled DNA.

The enzymatic incorporation of modified dNTPs into a growing DNA strand has intensively been studied. Modifications were detectable reporter groups such as digoxigenin or biotin, fluorochromes or aliphatic side chains covalently attached to the base. Incorporation efficiencies were determined with several DNA polymerases using linear primer-extension reactions followed by denaturing PAGE as a high-resolution detection system. We describe the enzymatic synthesis of DNA consisting of modified nucleotides exclusively. A defined template-primer system allows us to trace incorporation: (1) in up to 18 neighboring positions for several dUTP-derivatives; or (2) in stretches of DNA of up to 40 bases in length with complete substitution of all four natural dNTPs by differently modified counterparts. Synthesized DNA molecules are shown to particularly exhibit dramatically altered physico-chemical properties by contrast with native DNA. These results provide a fundamental data set for probe generation in single-molecule DNA sequencing (SMS).

Crystal structure of a DNA-dependent RNA polymerase (DNA primase).

Primases are essential components of the DNA replication apparatus in every organism. They catalyze the synthesis of oligoribonucleotides on single-stranded DNA, which subsequently serve as primers for the replicative DNA polymerases. In contrast to bacterial primases, the archaeal enzymes are closely related to their eukaryotic counterparts. We have solved the crystal structure of the catalytic primase subunit from the hyperthermophilic archaeon Pyrococcus furiosus at 2.3 A resolution by multiwavelength anomalous dispersion methods. The structure shows a two-domain arrangement with a novel zinc knuckle motif located in the primase (prim) domain. In this first structure of a complete protein of the archaeal/eukaryotic primase family, the arrangement of the catalytically active residues resembles the active sites of various DNA polymerases that are unrelated in fold.

Mineral and casein equilibria in milk: effects of added salts and calcium-chelating agents.

We have investigated the effects of adding a range of mineral salts and calcium-chelating agents on the distribution of casein and minerals between the non-pelleted and pelleted phases of milk obtained upon centrifugation at 78000 g for 90 min. Adding CaCl2 or mixtures of NaH2PO4 and Na2HPO4 to reconstituted skim milk (90 g milk solids/kg) at pH 6.65 increased both pelleted casein and pelleted calcium phosphate. Opposite effects were obtained by adding citrate or EDTA. The change in pelleted calcium phosphate was not simply related to casein release from the micelle. Upon adding 5 mmol EDTA/kg milk, 20% of the pelleted Ca, 22% of the pelleted phosphate and 5% of the micellar casein were removed. Increasing the concentration of EDTA to 10 mmol/kg milk decreased the pelleted Ca by 44% and the pelleted phosphate by 46%, and caused 30% of the micellar casein to be released. The effects of adding phosphate, citrate or EDTA at pH 6.65, followed by the addition of CaCl2, demonstrated the reversibility of the dissolution and formation of the micellar calcium phosphate. There were limits to this reversibility that were related to the amount of colloidal calcium phosphate removed from the casein micelles. Adding CaCl2 to milk containing > or = 20 mmol EDTA or > or = 30 mmol citrate/kg milk did not result in complete reformation of casein micelles. Light-scattering experiments confirmed that the dissolution of moderate amounts of colloidal calcium phosphate had little effect on micellar size and were reversible, while the dissolution of larger amounts of colloidal calcium phosphate resulted in large reductions in micellar size and was irreversible.

Interaction of cytochrome c with the blue copper proteins, plastocyanin and azurin.

Bimolecular rate constants have been determined for the reactions of native horse cytochrome c, eight 4-carboxy-2,6-dinitrophenyl (CDNP-) cytochromes c singly modified at lysines 7, 13, 25, 27, 60, 72, 86, or 87 and one 2,3,6-trinitrophenyl cytochrome c singly modified at lysine 13, with the blue copper proteins, plastocyanin (from parsley leaves) and azurin (from Pseudomonas aeruginosa). Plastocyanin, a protein having a negative charge of about -7, yields a bimolecular rate constant with native ferrocytochrome c of 1.5 x 10(6) M-1 S-1, which decreases with the modified cytochromes c to a minimum of 7.5 x 10(5) M-1 S-1 for the CDNP-lysine 13 derivative. Conversely azurin, a protein with an overall negative charge of only about -1 to -2, exhibits bimolecular rate constants with native ferrocytochrome c of 6.6 x 10(3) M-1 S-1 at pH 6.1 and 4.0 x 10(3) M-1 S-1 at pH 8.6, which increase upon modification of the cytochrome c to a maximum of 4.1 x 10(4) M-1 S-1 at pH 6.1 and 2.7 x 10(4) M-1 S-1 at pH 8.6, for the CDNP-cytochrome c modified at lysine 72. This behavior indicates that: 1) the reaction of cytochrome c occurs at a negatively charged site on plastocyanin, whereas azurin behaves as a positively charged reactant, the electrostatics governing to a large extent the relative reactivities of the modified cytochromes c; 2) in both cases the interaction domain on cytochrome c is located on the "front" surface of the protein and encompasses the solvent accessible edge of the heme prosthetic group, as is the case for all the reactions of cytochrome c with its mitochondrial protein redox partners, as well as for small inorganic redox complexes; and 3) the bimolecular rate constants for plastocyanin and azurin are orders of magnitude slower and the effects of lysine modifications far smaller than for the reactions with physiological systems, indicating that: (a) the electric fields generated by the reactants do not align them, prior to electron transfer, as effectively as for the physiological reaction partners of cytochrome c; and (b) there is an absence of a precise molecular fit between cytochrome c and the nonphysiological redox partners.